Characterization and evaluation of DOTA-conjugated Bombesin/RGD-antagonists for prostate cancer tumor imaging and therapy

Introduction: Here we present the metallation, characterization, in vivo and in vitro evaluations of dual-targeting, peptide-based radiopharmaceuticals with utility for imaging and potentially treating prostate tumors by virtue of their ability to target the α_Vβ₃ integrin or the gastrin releasing peptide receptor (GRPr). Methods: [RGD-Glu-6Ahx-RM2] (RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) was conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelator (BFCA) purified via reversed-phase high-performance liquid chromatography (RP-HPLC), characterized by electrospray ionization-mass spectrometry (ESI-MS), and radiolabeled with ¹¹¹In or ¹⁷⁷Lu. Natural-metallated compounds were assessed for binding affinity for the α_Vβ₃ integrin or GRPr in human glioblastoma U87-MG and prostate PC-3 cell lines and stability prior to in vivo evaluation in normal CF-1 mice and SCID mice xenografted with PC-3 cells. Results: Competitive displacement binding assays with PC-3 and U87-MG cells revealed high to moderate binding affinity for the GRPr or the α_Vβ₃ integrin (IC₅₀ range of 5.39±1.37 nM to 9.26±0.00 nM in PC-3 cells, and a range of 255±47 nM to 321±85 nM in U87-MG cells). Biodistribution studies indicated high tumor uptake in PC-3 tumor-bearing mice (average of 7.40±0.53% ID/g at 1h post-intravenous injection) and prolonged retention of tracer (mean of 4.41±0.91% ID/g at 24h post-intravenous injection). Blocking assays corroborated the specificity of radioconjugates for each target. Micro-single photon emission computed tomography (microSPECT) confirmed favorable radiouptake profiles in xenografted mice at 20h post-injection. Conclusions: [RGD-Glu-[¹¹¹In-DO3A]-6-Ahx-RM2] and [RGD-Glu-[¹⁷⁷Lu- DO3A]-6-Ahx-RM2] show favorable pharmacokinetic and radiouptake profiles, meriting continued evaluation for molecular imaging in murine U87-MG/PC-3 xenograft models and radiotherapy studies with ¹⁷⁷Lu and ⁹⁰Y conjugates. Advances in Knowledge and Implications for Patient Care: These heterovalent, peptide-targeting ligands perform comparably with many mono- and multivalent conjugates with the potential benefit of increased sensitivity for detecting cancer cells exhibiting differential expression of target receptors.